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A site-directed multi-site gene mutagenesis method

A gene-directed mutation and multi-site mutation technology, applied in the field of genetic engineering, can solve the problems of high temperature resistance, complicated recombinant enzyme purification process, high requirements for transportation and storage methods, and achieve the effect of simple operation process and low cost

Inactive Publication Date: 2016-03-16
HUAQIAO UNIVERSITY
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Problems solved by technology

However, the purification process of recombinant enzymes is relatively complicated, it is not resistant to high temperature, and the requirements for transportation and storage methods are relatively high.

Method used

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  • A site-directed multi-site gene mutagenesis method
  • A site-directed multi-site gene mutagenesis method
  • A site-directed multi-site gene mutagenesis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 1. Preparation of HU protein

[0027] 1.1) Insert HU-α or HU-β subunits or dimers of HU-α and HU-β from Escherichia coli into the pET22 expression vector, transform by ligation and verify by colony PCR, enzyme digestion and sequencing , the recombinant expression vector pET22-Hu-α or pET22-Hu-β or the dimer of Hu-α and Hu-β can be obtained;

[0028] 1.2) Use Escherichia coli competent cells BL21 to transform the constructed recombinant vector pET22-HU-α or pET22-HU-β or the dimer of HU-α and HU-β, and take the positive strain containing the recombinant plasmid to add Cultivate in LB liquid medium overnight, then inoculate the bacterial solution into 50ml of LB medium at a ratio of 1%, and culture it in a shaker at 37°C until the OD value is 0.6-0.8; add IPTG, and place the bacterial solution at 37°C Induce expression in a shaker for 3.5h;

[0029] 1.3) Take the bacterial liquid after induction of expression and centrifuge to obtain the bacterial cells, lyse and releas...

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Abstract

A site-directed multi-site gene mutagenesis method is provided. The method includes a step of designing primers containing mutagenesis sites according to base positions of site-directed gene mutagenesis, namely a step of introducing the mutagenesis sites to overlapping regions through primer designing, a step of preparing PCR segments containing the mutagenesis sites by utilization of PCR amplification, and a step of splicing the segments in a seamless manner by utilization of non-specific-DNA-bonded protein HU in place of recombinase, thus achieving site-directed multi-site mutagenesis.

Description

【Technical field】 [0001] The invention relates to the technical field of genetic engineering, in particular to a method for gene-directed multi-site mutation. 【Background technique】 [0002] Gene mutation refers to abnormal changes in the structure, replication or phenotype function of individual dNMP (deoxynucleoside monophosphate) residues or fragments of DNA, also known as DNA damage. In vitro site-directed mutagenesis is an important experimental method in various fields of biology and medicine. It is mostly used to transform, optimize target genes, and explore the regulatory sites of promoters. It is also an effective means for studying the complex relationship between protein structure and function. powerful tool. And gene mutation is the fundamental source of biological variation, providing raw materials for biological evolution. Genetic recombination can produce a variety of offspring with a variety of gene combinations, and some of the offspring will contain gene ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/102
Inventor 戚智青唐黎侯丹刁勇
Owner HUAQIAO UNIVERSITY
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